Department of Physiology and Biophysics, University of California, Irvine, California, USA.

Abstract

A central feature of the lipid raft concept is the formation of cholesterol-rich lipid domains. The introduction of relatively rigid cholesterol molecules into fluid liquid-disordered (L(d)) phospholipid bilayers can produce liquid-ordered (L(o)) mixtures in which the rigidity of cholesterol causes partial ordering of the flexible hydrocarbon acyl chains of the phospholipids. Several lines of evidence support this concept, but direct structural information about L(o) membranes is lacking. Here we present the structure of L(o) membranes formed from cholesterol and dioleoylphosphatidylcholine (DOPC). Specific deuteration of the DOPC acyl-chain methyl groups and neutron diffraction measurements reveal an extraordinary disorder of the acyl chains of neat L(d) DOPC bilayers. The disorder is so great that >20% of the methyl groups are in intimate contact with water in the bilayer interface. The ordering of the DOPC acyl chains by cholesterol leads to retraction of the methyl groups away from the interface. Molecular dynamics simulations based on experimental systems reveal asymmetric transbilayer distributions of the methyl groups associated with each bilayer leaflet.

Transbilayer distribution of terminal methyl groups and water in a liquid-disordered DOPC bilayer. (A) Scattering-length density profiles on an absolute scale along the bilayer normal of DOPC bilayers. Temperature: 21°C. The solid black line shows the reference bilayer profile determined using nondeuterated DOPC hydrated at 66% relative humidity (RH) (5.4 waters per lipid) with H2O. Blue-shade profiles are for DOPC bilayers hydrated at 66% RH in a series of D2O/H2O mixtures (10, 20, and 50 mol % D2O). Red-shaded profiles correspond to bilayers formed from mixtures of nondeuterated DOPC and terminal-methyl deuterated DOPC (d6-DOPC) (49, 73, and 97 mol % d6-DOPC). The density profile amplitudes are presented in units of scattering-length per unit length, corresponding to the scattering-length density of a unit cell multiplied by the area per lipid (S; see ). The X axis shows the distance from the bilayer center (z) with zero positioned in the middle of the bilayer. Space-filling representations of disordered DOPC molecules are shown above the panel (oxygen, red; carbon, gray; nitrogen, blue; phosphorous, gold; hydrogen, white; deuterium, orange). (B) Difference scattering-length density profiles obtained by subtracting the reference profile (black curve, panel A) from the profiles determined with deuterated water (blue-shaded curves, panel A) and deuterated terminal methyl groups (red-shaded curves, panel A). The difference profiles use the same coloring scheme as in panel A. These data reveal the extreme disorder of liquid-disordered bilayers. The outer wings of the CH3 groups overlap the water distributions and account for ∼20% of the total number of CH3 groups (). Similar results were obtained at 86% and 93% RH (7.7 and 9.4 waters per lipid, respectively; ). The estimated maximum uncertainty for all profiles is indicated by the black error bar determined from error bands computed by the method of Wiener and White (). Examples of such error bands are shown in . To reduce the complexity of the plots, the bands were not included here; the same data with complete error bands are shown in .

Transbilayer distribution of terminal methyl groups in a liquid-ordered DOPC/cholesterol (2:1) bilayer. Scattering-length density profiles on an absolute scale along the bilayer normal of DOPC and 2:1 DOPC/cholesterol bilayers. Temperature: 21°C. Hydration: 86% RH. (A) The dashed blue curve is the reference profile for a 2:1 DOPC/cholesterol bilayer, and the solid blue curve is for 2:1 d6-DOPC/d6-cholesterol (cholesterol-2,2,3,4,4,6-d6). The dashed red curve is the reference profile for DOPC, and the solid red curve is for 2.7:1 d6-DOPC/DOPC (i.e., 73 mol % d6-DOPC). Space-filling representations of disordered DOPC molecules (light red background) and cholesterol molecules (light blue background) are shown above the panel (red, oxygen; gray, carbon; blue, nitrogen; gold, phosphorous; white, hydrogen; orange, deuterium). (B) Difference scattering-length density profiles obtained by subtracting the reference profiles from the profiles determined using d6-DOPC (yellow curve surrounded by red band) and d6-DOPC/d6-cholesterol (blue curve surrounded by lighter blue band). The d6-DOPC curve shows that the terminal methyl groups have a broad transbilayer distribution similar to the distribution observed at 66% RH (B). The broad red band represents estimates of experimental uncertainty computed using the methods of Wiener and White (). The d6-DOPC methyl peak has been rescaled to account for the lower d6-DOPC concentration (73 mol %) in the DOPC bilayers compared to the d6-DOPC/d6-cholesterol bilayers, which were made with 100% d6-DOPC. The d6-DOPC/d6-cholesterol curve shows that the cholesterol causes the terminal methyl groups to retract from the interface. The pair of peaks located at ∼±15 Å relative to the bilayer center are due to the six deuterium atoms on the hydroxyl-containing A ring of cholesterol. The broad light-blue band represents estimates of experimental uncertainty computed according to the methods of Wiener and White (). (C) Difference scattering-length density profiles for d6-DOPC/cholesterol (determined independently from the data presented in A and B) obtained by subtracting a reference 2:1 DOPC/cholesterol profile from the d6-DOPC/cholesterol (blue curve surrounded by lighter blue band). The d6-DOPC difference structure of panel B is included for comparison. The dotted blue curves are Gaussian-fit curves of the deuterated A ring of the d6-DOPC/d6-cholesterol difference structure of panel B. The repeat distances were determined to be 50.4 ± 0.1 for neat DOPC bilayers and 52.7 ± 0.2 for 2:1 DOPC/cholesterol, respectively. The analysis was performed considering nw = 7.7 water molecules per unit cell for neat DOPC () and nw = 7.9 water for 2:1 DOPC/cholesterol. The latter value was determined by using the terminal methyl of d6-DOPC peak for calibration, which allows determination of the absolute amplitude of the water peak ().

Simulated transbilayer distributions of terminal methyl groups in liquid-disordered and -ordered DOPC/cholesterol (2:1) bilayers. (A) Snapshot of the all-atom MD simulation of DOPC. The terminal methyl groups are represented as spheres and colored purple and magenta to distinguish the methyls of each leaflet. Acyl chains shown in stick representation are gray. Phosphatidylcholine headgroups are also shown in stick representation and colored according to the element (red, oxygen; blue, nitrogen; gold, phosphorous; white, hydrogen). Water is represented by the aquamarine bands. The simulation box contained 288 DOPC and 2276 water molecules. The d-spacing under NPT conditions was 48.6 ± 0.4 Å. (B) Transbilayer distribution of terminal methyls for DOPC observed in the all-atom MD simulations of the experimental results described in . The solid aquamarine curve shows the distribution for all terminal methyl groups. The broad wings observed experimentally are also observed in the simulation. The maroon and purple curves show the distributions of methyl groups associated with the left and right bilayer leaflets, respectively. (C) Snapshot from the all-atom MD 2:1 DOPC/cholesterol. Cholesterol molecules, colored goldenrod, are shown in stick representation. As observed experimentally, the bilayer is thicker than in panel A due to the ordering effect of the cholesterol molecules on the DOPC molecules. The simulation box contained 192 DOPC, 96 cholesterol, and 2285 water molecules. The d-spacing under NPT conditions was 53.1 ± 0.7 Å. (D) Transbilayer distribution of terminal methyls for DOPC in 2:1 DOPC/cholesterol bilayers observed in all-atom MD simulations of the experimental results described in . The solid aquamarine curve shows the distribution for all terminal methyl groups. As in the experiments, the DOPC methyls retract from the membrane interface in the presence of cholesterol. The maroon and purple curves show the distributions of methyl groups associated with the left and right bilayer leaflets, respectively.

Order-parameter profiles and fraction of gauche conformations for DOPC acyl chains. (A) Order-parameter profiles for the acyl chains of DOPC in the absence and presence cholesterol (2:1 DOPC/cholesterol) determined from MD simulations. The orientational order parameters SCD of the DOPC oleoyl chains (sn-1 and sn-2) were calculated from the orientation of the C-H bonds of each methylene position relative to the bilayer normal. Specifically, SCD = |<P2(cosθ)>|, where P2 is the second Legendre polynomial, and θ is the angle between a C-H bond and the bilayer normal. (B) Fraction of gauche conformations along the DOPC sn-1 and sn-2 chains in the absence and presence of cholesterol (2:1 DOPC/cholesterol) determined by MD simulations. The fraction of gauche conformations was calculated for the DOPC and DOPC/cholesterol systems from the dihedral angle distributions along the sn-1 and sn-2 DOPC oleoyl chains. The data show that the presence of cholesterol has little effect on the distributions of gauche conformations.